Diagnosing and uncovering the genetic basis of disease has been revolutionized by whole-exome sequencing (WES), allowing discovery of new disease genes and improving the rate of clinical diagnosis for rare genetic conditions. Indeed, the genetic basis of childhood disorders can be identified in approximately 25% of clinical cases, where successful molecular diagnosis frequently has a major impact on patient management and treatment[1, 2]. Prioritization of candidate variants in the remaining cases remains challenging due mainly to insufficient understanding of the functional consequence of substantial fraction of candidate variants[3]. Large scale functional characterization of genomic variation by simultaneously sequencing RNA from the patient can reveal genotype-phenotype correlation, can highlight gene expression profile that is associated with the studied genetic condition, and allows immediate evaluation of in silico prediction algorithms to the effect genomic variants have on gene expression, alternative splicing, exon usage, gene fusions[4]. In breast and pancreatic cancer integrated analysis of DNA and RNA has been successfully utilized to obtain insight into molecular mechanisms that explain pathogenicity and uncovered potential therapeutic targets to improve patient management[5-7]. In addition, sequencing RNA (RNAseq) has been utilized in the context of the effect epigenetic modifications have on gene expression [8, 9]. Integrative analysis of whole-exome and RNA sequencing data in X-linked disorders may also be informative both in diagnosis and gene discovery for phenotypes emerging due to epigenetic changes such as X chromosome inactivation[10].
In the process of X-chromosome inactivation (XCI), in females, cells undergo epigenetic inactivation of one of the inherited, parental X chromosomes resulting in consecutive daughter cells expressing one X [11, 12]. The proportion of cells with either parental X as the active is defined by the XCI ratio that ranges from 50:50 random to 100:0 completely skewed. Epigenetic analysis of X chromosome in unaffected females indicate that XCI ratio varies in the general population and is normally distributed [13]. Although, on the cellular level X-linked alleles are expressed in a dominant fashion due to XCI, in cell populations they show mosaic pattern which can lead to heterogeneous phenotype in females who are carriers for disease causing, deleterious mutations[14].
In X-linked neurological disease, mode and magnitude of XCI can influence disease severity and outcome [15]. Indeed, case-control studies demonstrate that skewed XCI is common among females who are carriers for X-linked Mental Retardation disorders (XMLR)[16]. XCI may also lead to asymptomatic carrier status by selective advantage of cells expressing the wild-type alleles[17]. One of the difficulties diagnosing females with X-linked diseases and skewed XCI is the broad and overlapping description of clinical phenotype, the limited availability of similar cases, and lack of high-throughput expression-based methods to estimate XCI[15]. Routine, clinical method to estimate XCI ratio rely on the HUMARA differential DNA methylation assay that targets a polymorphic short tandem repeat (STR) in the human androgen receptor gene (AR)[18]. Methylation of this repeat is associated with X chromosome inactivation. Although >90% females are polymorphic at this site to differentiate between the two chromosome copies, it provides expression information indirectly from DNA, and, relies on a single locus[13]. There is also conflicting evidence whether DNA methylation can reflect the quantitative expression ratio of active and inactive X with high accuracy when compared to direct allelic expression-based methods[19, 20].
Thus, a need exist for an improved processes for scanning for and analyzing these X-linked variations and simultaneously obtaining functional implications of those genomic variations.